Advances in nucleic acid research during the past decade have considerably simplified the assays which utilize DNA and RNA probes for research and diagnostic purposes. Among various assays, the recently introduced modification of the gene amplification method called polymerase chain reaction (PCR) qualifies as a technological breakthrough. PCR in an in vitro gene amplification method whereby the DNA from a selected region of a genome can be amplified by more than a million-fold in a few hours, provided that at least a portion of its nucleotide sequences are already known. Segments of the gene sequence that are at both sides of the portion of the gene which one wishes to amplify, are usually synthesized by an automatic DNA synthesizer. These two oligonucleotides, called "primers", are usually 10-30 base pairs (bp) long. The primers hybridize to opposite strands of DNA, and flank the region of interest in the target DNA.
The PCR method has advantages over serological testing. For example, PCR has been used to detect HIV-1 exposure in the seronegative sexual partners of HIV-1-seropositive individuals, in HIV-1-seronegative infants and children, and in health care workers accidently exposed to HIV-1-positive blood or body fluids.
There are several disadvantages in the conventional DNA-PCR method. Due to the sensitivity of the method, a small amount of contamination (usually from DNA fragments aerosolized during a previously performed amplification) may cause a false positive result. Current methods also do not permit determination of which cell types carry a certain gene.
The ability to identify individual cells, either latently or productively infected, under the microscope would be extremely useful in delineating a latent state and emergence from it. This would be useful, not only in understanding the development of infection, but also as a more direct quantitative measure of the effects of antiviral therapy, and as an aid in understanding the mechanism of transmission of HIV-1.
Since the first description of human immunodeficiency virus type I (HIV-1) as the etiologic agent of AIDS, the numbers of cells infected in vivo with HIV-1 isolated from individuals at various clinical stages of disease have been evaluated. These studies sought to correlate levels of HIV-1 with disease pathogenesis and to determine the clinical course of HIV-1-seropositive individuals.
The most detrimental clinical consequence of infection with HIV-1 is the severe depletion of CD4-positive lymphocytes. It was assumed that such depletion was the result of selective infection and destruction of CD4-positive lymphocytes by HIV-1. The studies of Harper et al., Proc. Natl. Acad. Sci. USA, 83:772-6 (1986), demonstrated that by using in situ hybridization for HIV-1-specific RNA, one can identify only 1:10,000 to 1:100,000 peripheral blood mononuclear cells (PBMC) and lymph node cells positive for HIV-1 in vivo. These studies did not discount the possibility that HIV-1 may be present in a latent proviral form not expressing viral mRNA. The teaching of Harper et al. did not eliminate the possibility that HIV-1 selectively expressed only low levels of multiply-spliced RNA and did not express unspliced genomic HIV-1 RNA. The findings of Harper et al. taken with observations that the rate of isolation of HIV-1 from infected individuals was low, suggested that indirect mechanisms might mediate HIV-1-induced CD4-positive lymphocyte destruction. Several mechanisms have been proposed to account for the severe depletion of CD4-positive lymphocytes including: (1) production of direct cytopathic effect of HIV-1 on CD4-positive cells; (2) generation of HIV-1-specific cytotoxic T-lymphocytes (CTL) or antibody-dependent cellular cytotoxicity (ADCC), which destroy cells expressing surface HIV-1-specific proteins; (3) generation of giant cell syncytia formation, secondary to an interaction of the CD4 receptor and a fusion domain of the HIV-1 envelope glycoproteins; and/or (4) production of antibodies against T lymphocytes, bone marrow stem cells or immature thymocytes.
Recent data suggest a higher level of PBMC containing HIV-1-specific RNA in infected individuals. Studies utilizing limiting dilution assays have shown that infectious HIV-1 can be isolated from an average of 1 in 400 PBMC obtained from patients with AIDS, however, higher viral levels have been detected during acute HIV-1 seroconversion.
Since the introduction of Taq, the thermostable polymerase which brought convenience to the polymerase chain reaction (PCR) method, special attention has been directed to the study of HIV-1 infection using this method. Modifications of the PCR method have been used to quantitatively or semi-quantitatively assess the relative frequencies of HIV-1-infected cells in PBMC, lymph nodes and other cell types. Schnittman et al., Science, 245:305-8 (1989); Ann. Int. Med., 113:438-43 (1990), disclose using a combination of cell sorting and quantitative DNA-PCR techniques to observe that at least 1% of CD4-positive lymphocytes in patients with AIDS are infected with HIV-1. For patients who are HIV-1 asymptomatic, the levels of CD-4-positive lymphocytes was shown to be between 1:100 and 1:100,000.
Spector et al., J. Infect. Dis., 164:4703-5 (1991), disclose using a "booster" PCR method and have calculated that at least 10% of CD4-positive lymphocytes carry HIV-1 provirus in AIDS and symptomatic HIV-1-infected patients, whereas a relatively lower proportion of CD4-positive lymphocytes are positive in HIV-1-seropositive individuals.
Studies utilizing a quantitative DNA PCR technique have suggested that there is a correlation between the clinical stage of HIV-1 infection and the level of HIV-1-specific PCR signals.
A current limitation to PCR methodologies utilizing isolated DNA is that one cannot directly associate the amplification results to a specific cell type or easily measure the percentage of cells which carry the target sequence. The HIV-1 virus has been demonstrated to infect CD4-positive lymphocytes, CDS-positive lymphocytes, monocytes, fibroblasts and glial cells in vivo. The CD4-positive lymphocyte is believed to be the primary reservoir for HIV-1 in the bloodstream and cells of the monocyte/macrophage lineage are believed to be the major virus reservoir in solid tissues. Therefore, it is highly desirable to identify all cell types which carry the virus in vivo and determine which cells actively produce HIV-1.
Haase et al., Proc. Natl. Acad. Sci. USA, 87:4971-5 (1990), disclose the development of an in situ PCR methodology for Visna virus, a viral pathogen of sheep. Moreover, Haase et al. teach in situ PCR performed on cells infected with Visna virus in suspension. Haase et al. further disclose that the reactions are performed using a Perkin-Elmer/Cetus DNA thermal cycler. Following the PCR reaction in solution, the contents of the tubes were centrifuged, resuspended and applied to a slide. The PCR-amplified nucleic acid was fixed to the slide and hybridized to .sup.125 I-labelled viral DNA for detection.
Oakes et al. (EP388171) teach purifying single-stranded targeted nucleic acid using non-porous, non-magnetic particles with complementary nucleic acid attached. Oakes et al. claim a method using standard PCR hybridization methodology and include a step of separating the hybrid from the remainder of the specimen with a non-porous, non-magnetic particle. Oakes etal. indicate that the invention is useful since purified nucleic acids can be rapidly and simply purified. Once purified, the hybrids can be amplified by polymerase chain reaction.
Wang et al. (WO9102817) claim a method for quantifying a target nucleic acid segment in a sample. Wang et al. indicates that the invention is useful for determining the quantity of specific RNA molecules in a biological sample.
Gyllensten and Erlich (WO9003444) teach a method for generating single-stranded DNA by PCR that can be linked to an automatic sequence system for rapid sequence determination. The production of single-stranded PCR products using limiting concentrations of one of the two primers is also disclosed.
Innis (WO9003443) teaches a method for structure-independent amplification of DNA by PCR using structure-destabilizing base analog in the amplification reaction. Innis further teaches that the method is useful to increase the specificity of PCR on nucleic acid templates that contain secondary structure and/or compressed regions.
Manos et al. (WO9002821) teach detecting human papilloma-virus using consensus primers in PCR to amplify particular genomic regions. The patent discloses a PCR reaction using at least a pair of primers complementary to separate strands of HPV DNA which hybridize to it and produce an extension product.
Post et al. (WO9001547) teach isolation of thymidine kinase encoding DNA from herpes virus from degenerate primers and the production of thymidine kinase negative feline herpes virus used to produce a live vaccine.
Erlich et al. (WO8911547) disclose HLADP genotyping by amplifying target DNA then hybridizing the amplified target to a panel of sequence-specific oligonucleotides. It is disclosed that the method is especially useful for assessing the risk of autoimmune disease.
U.S. Pat. No. 5,008,182 (Sninsky et al.) discloses the detection of AIDS-associated virus by PCR in free solution or after immobilization on a solid support.
Until the present invention, there have been no methods which allow in situ PCR amplification of specific DNA fragments in intact cells.